1. To define periods in the maturation of dopamine neurons when they are sensitive to different external survival signals.[unreadable] [unreadable] We have defined a period of naturally occurring death during the first 10 days when midbrian dopamine (DA) neurons differentiate in vitro. This assay has identified survival mechanisms specific to the class of dopamine neuron at most risk in Parkinsons disease. Many neurotrophic factors have been reported to affect survival of DA neurons but the pathways that integrate the effects of thee different signals are not defined. Based on our studies of hippocampal neurons showing that different survival signals act at different stages of differentiation. We are now using this developmental approach to define the signaling logic that controls the survival of dopamine neurons. [unreadable] [unreadable] 2. The role of foxa2 in the specification of dopamine neurons. [unreadable] [unreadable] We have mapped the origins of dopamine neurons to the floor plate, a specialized structure previously thought to function only transiently during development. The clearest evidence comes from a genetic tracing experiment that unequivocally shows tyrosine hydroxylase positive dopamine neurons are derived from cells that expressed SHH. The floor plate is specified by the early expression of the transcription factors foxa2 and lmx1b. Foxa2 interacts closely with shh and we have shown using foxa2 null embryos and by expressing foxa2 in stem cells, that foxa2 function is necessary for dopamine neuron differentiation. In contrast to the importance of foxa2, when we looked closely at SHH function in vitro, there was no evidence for a morphogenic effect on dopamine neuron precursors. Further experiments will define the role of foxa2 to specify or re-specify precursors to generate dopamine neurons.[unreadable] [unreadable] 3. The role of foxa2 in the survival of adult dopamine neurons. [unreadable] [unreadable] Animals that have only one copy of the foxa2 gene show spontaneous loss of dopamine neurons with age. This mouse will likely be very widely used because although many mutations have been identified that contribute to Parkinsons disease and related disorders, it has been difficult to reproduce the specific loss of dopamine neurons in an animal model. This animal has the potential to teach us a number of important lessons: why some dopamine neurons are more at risk than others, why the disease is progressive and why dopamine neurons are sensitive to mutations in genes that are widely expressed? [unreadable] [unreadable] Much of what is known about the relationship between the various symptoms in Parkinsons patients and the underlying defects in the nigrostriatal system comes from lesion studies in the rat. The foxa2 heterozygous mouse allows us to study for the first time, in an animal model, motor and cognitive behaviors in the context of a progressive neurodegenerative process. We have performed basic studies to demonstrate motor deficits in severely affected old foxa2 mutant mice. In foxa2 mutant mice that have not yet developed motor deficits, we propose to test for behavioral deficits related to those that have been observed in Parkinsons patients and in rat lesion models. These behavioral tests together with measurement of striatal dopamine by HPLC and labeling of dopaminergic innervation will give us insight on the role of specific striatal regions in controlling cognitive functions and may reveal compensatory mechanisms regulating these behaviors in the mutant mice. [unreadable] [unreadable] 3. Human Genetics and the biology of dopamine neurons.[unreadable] [unreadable] In collaboration with Jeff Vances group (Duke University and now at the University of Miami) we have identified single nucleotide polymorphisms (SNPs) in the FGF20 gene associated with Parkinsons disease. We continue to study the role of genetic polymorphisms in the FGF20 in humans. The polymorphism that contributes to an elevated risk of PD is common in both North America and Japan. This allows the effect of this gene to be assessed in existing cognitive and anatomical databases. We will ask if the risk allele is associated with impaired cognitive abilities and alterations in brain structure. [unreadable] [unreadable] 4. Supporting dopamine neuron survival in vivo.[unreadable] [unreadable] We have shown that a single intra-ventricular injection of Notch ligands alone or in combination with other angiogenic factors promotes widespread activation of the stem cell niche in the adult brain and rescues dopaminergic neurons in a model of PD. These data suggest vascular cytokines promote regenerative responses to brain injury. A major goal of our group is to set up in vitro assays that predict the activation of the stem cell compartment in vivo. In this project, we will use our growing understanding of survival signaling in the stem cell niche to rescue injured dopamine neurons in vivo. This approach may allow a more rapid transition to clinical application than cell replacement therapy.